This invention relates to a device for examining an object in three dimensions.
In confocal microscopy, an object is illuminated in known fashion through an aperture diaphragm and the illuminated point is observed by a radiation detector for which the light-sensitive surface is just as small as the illuminated point (Minsky, M., U.S. Pat. No. 3,013,467 and Minsky, M., Memoir on inventing the confocal scanning microscope. Scanning 10, p. 128-138). Compared to conventional microscopy, confocal microscopy has the advantage of delivering resolution in depth (measurement of the z axis) and of creating little scattered light during imaging. Only the plane of the object in focus is brightly illuminated. Object planes above or below the focus plane receive much less light. The image is built up through a scanning process. One or more points may be illuminated and observed simultaneously.
Three scanning methods are well known: mirror scanning, Nipkow disk, and electronic scanning using a matrix detector. Additional details on prior art relating to scanning with a mirror or Nipkow disk may be found in the Handbook of Biological Confocal Microscopy, Plenum Press, New York (James D. Pawley, Editor).
A confocal imaging system with confocal illumination through an aperture plate and electronic scanning by a matrix detector was first proposed in DE 40 35 799. A matrix detector is employed here in which the pixels are light sensitive only one a portion (30%) of the surface assigned to the pixel, and on the illumination side, an aperture plate is typically used which has the same number of holes as the imaging sensor has light-sensitive pixels. The information in depth is gained by recording multiple images from different focus planes and individually evaluating the brightness maximum for the different pixels in a computer.
Document DE 196 48 316 describes an arrangement which is typically provided with one illumination hole on the aperture plate for every four detector pixels assigned to it, and with a prism array immediately in front of the matrix detector. The prism array acts as a beam-forming element which splits the light of each illumination point such that two crescent images are formed outside the focus. Document DE 196 51 667 A1 describes an arrangement in which likewise typically one illumination hole on the aperture plate is assigned to four detector pixels each and which contains an array of anamorphote lenses immediately in front of the detector array. One lens is assigned to each illumination hole. Here the anamorphote lenses also act as beam-forming elements producing an image of the illumination point, the image being circular in focus and oval outside of focus. In these last two arrangements, the information in depth is gained by evaluating the difference between light signals of adjacent pixels.
Arrangements DE 40 35 799, DE 196 48 316 and DE 196 51 667 A1 have the advantage, among others, that many measurement points in depth may be recorded simultaneously, yet have the disadvantage that color images cannot be recorded. The object of the present invention is therefore to disclose an approach by which images may be recorded confocally using available color-capable matrix detectors. This requirement is found for example, in genetic technology, cancer research and cancer screening where there is a need within a short period to scan many tissue cells for small (e.g. 200 nm) fluorescing or dyed sites in three dimensions.
The invention provides for arranging one aperture plate each, both on the illumination side and on the observation side, in those planes which are optically conjugate with the focus plane of the object and arranging at a suitable distance a color-capable matrix detector behind the aperture plate on the observation side, i.e. outside of focus.
The diagrams show examples of possible practical embodiments of the invention.